Type 1 diabetes (T1D) is a debilitating autoimmune disease with the incidence on the increase in developed countries. Many facts point at microbial involvement in the regulation of T1D. In preliminary studies we have established that: a. non-obese diabetic (NOD) mice lacking MyD88 adaptor were resistant to spontaneous T1D when housed in conventional specific-pathogen-free (SPF) conditions; b. MyD88 KO NOD mice did not show systemic tolerance to islet antigens, but did show tolerance of T cells to such antigens in the local pancreatic lymph nodes; c. when the same animals were made germ-free (GF), they developed diabetes with the frequency of about 100%; d. when GF MyD88 KO animals were reconstituted with the defined microflora, the diabetes incidence was reduced. Taken together the data suggested that commensal (not pathogenic) microbes in the gut influenced the development of T1D. The likely role of MyD88 is to control commensal microbes. When MyD88 is missing, the changes in gut microbiota ensue. To support that idea, we have performed the metagenomic analysis of the gut microbiota (16S rRNA genes sequencing) from normal NOD and MyD88 KO animals, and discovered significant and meaningful differences between them. Taking advantage of knock-out and conditional knock- out mice, the germ-free technology and metagenomic sequencing approach, we will pursue the following aims 1. Uncover MyD88-dependent mechanisms that control commensal microbes, thus promoting T1D. We will test a possible role of TLR2 as well as the role of B cells and intestinal epithelial cells in control of intestinal microbiota and study the cellular basis for resistance of MyD88 KO mice to T1D by conditional elimination of MyD88. 2. Elucidate MyD88-independent mechanisms by which commensals cause T cell tolerance. We will test different Pattern Recognition receptors (PRRs) for their role in prevention of T1D. We will use the results of a gene expression analysis: test expression of identified genes in cellular subsets in PLN and an importance of CCR5 in prevention of T1D by microbiota. We will address the role of antigen presenting cells (APC) and regulatory T cells (Treg) in local tolerance induced by microbiota. 3. Study the role of intestinal microbiota in prevention of T1D. We will test a hypothesis that particular bacteria protect against T1D and test the ability of selected microbial communities to reverse clinical diabetes in GF mice by themselves or in combination with immunosuppression. PUBLIC HEALTH RELEVANCE: The proposal is devoted to the studies of animals (NOD, non-obese diabetic strain of mice) prone to Type 1 diabetes (T1D) and also lacking control over intestinal microbes due to the disruption of their innate immune system. These mice are protected from T1D by their intestinal microbes, whereas same animals without microbes (germ-free, GF) are not protected. Using these animals and their GF counterparts, we will dissect the signaling pathways involved in protection from T1D, characterize microbial lineages capable of protection from T1D, and perform microbial transplants to test the possibility of translation of our prevention tactics to humans.